The long-term objectives of the proposed research are to understand the complex dialogue that occurs between humans and their microbiota during the initial minutes to hours of their interaction. These relationships, whether pathogenic or beneficial, typically begin at mucosal surfaces, sites that are often inaccessible to experimental study. In addition, they usual support a complex consortium consisting of hundreds of microbial species. At these sites, the interplay between the surface features of bacteria and the corresponding mechanisms of host recognition controls the initiation and persistence of both beneficial and pathogenic associations. How is this same cellular and molecular 'language' used differently to produce the two opposite outcomes of health or disease? To answer this question in the face of the daunting microbial complexity in the human body, simpler model systems are used that are more amenable to experimental manipulation. Biologists use such systems to discover fundamental principles that reveal how an individual bacterial species associates successfully with its host. One such model is the binary symbiosis between the bioluminescent Gram-negative bacterium, Vibrio fischeri, and its squid host, Euprymna scolopes. As in humans, the squid's relationship with this bacterium begins a new each generation, with the microbe transitioning from the environment into a functional beneficial relationship. In both cases, a 'winnowing' occurs that promotes an association with only that small subset of environmental bacteria that are recognized through complex molecular interactions. In the squid-vibrio system, this process occurs over minutes to hours, and can be directly imaged in its entirety. As such, the system offers the rare opportunity to decipher, with high temporal and spatial resolution, the reciprocal molecular and biochemical dialogue that is essential for a natural association to develop normally. This project brings together a team of scientists with expertise in cell and developmental biology, biochemistry, and genetics, to address the following specific aims: 1) to examine how interactions between host cells and the specific symbiont shape the microenvironment to promote partner recognition and specificity; 2) to characterize the genetic factors of the symbiont that are essential to the selection process; and, 3) to define how bacterial motility and outer membrane vesicles, well known virulence and colonization determinants, play a key role in the dialogue between host and symbiont.